Characterization of subtilosin gene in wild type Bacillus spp. and possible physiological role

In a designed study to screen for antimicrobial exhibiting bacteria using molecular aspects, Bacillus species were considered to investigate antibiotic biosynthesis genes. 28 bacterial strains and 3 induced mutants were screened for the presence of subtilosin gene (sbo) and subtilosin through PCR and Mass spectrometry respectively. Sbo gene was detected in 16 out of 28 Bacillus strains. The results from gene sequences deliberated by multiple sequence alignments revealed high-level homology to the sequences of the sbo-alb gene locus of B. subtilis 168 and the other limited reported strains. Hence, this report provided additional strains to support the idea of subtilosin gene predominance amongst Bacillus strains isolated from environment and to find different species containing homologous genes, furthermore the utilization of its conserved region as a means of identifying Bacillus spp. that produce subtilosin. This is the first report to confirm the detection of subtilosin production from B. amyloliquefaciens.

Bacterial identification. Identification of the isolated strains was carried on by sequence homology of 16S rDNA accompanied by morphological and biochemical characterization. Identification to the species level was defined as a 16S rDNA sequence similarity of ≥ 99% with that of the prototype strain sequence in Gen-Bank; identification at the genus level was defined as a 16S rDNA sequence similarity of ≥ 97% with that of the prototype strain sequence in GenBank. The biochemical profile of test isolates was determined with the API 50 CHB strips following the manufacturer's instructions (bioMerieux, France). This test allows bacterial strains to be classified according to their ability to ferment 49 different carbohydrates. The results were analyzed with the APILAB Plus software (bioMerieux, France).
Antibiotic assay. Samples of culture supernatant containing the antibiotic checked for activity using an agar-well diffusion assay 33 . Fifty µL of Bacillus fusiformis liquid culture of 0.3 OD 600 was spread onto the surface of Petri dish containing L-agar. 50 µL antibiotic sample was transferred into the well made in media plates using a sterile cork borer. The sample was allowed to diffuse into the agar and the plate was inverted and incubated at 37 °C until a lawn of the indicator bacteria appeared on the plate (approximately 10-16 h). DNA isolation, extraction and PCR. Genomic DNA extracted from overnight-inoculated bacterial culture in N-broth at 37 °C with 120 rpm. The extraction carried out using gene extraction kit (Biorad). PCR

Production of (-A) No activity mutants.
Mutants from strain-7 a confirmed producer of subtilosin were generated from exposed culture to UV light for different intervals. The surviving bacteria were screened for the disruptions in sbo gene through PCR and subtilosin production by MALDI-TOF-MS.

MALDI-TOF-mass spectrometry. Fractions correlated with Subtilosin A from TLC and Reverse phase
HPLC were analyzed using MALDI-TOF-MS. 2 µL of sample mixed with 2 µL matrix solution (2 mg of alphahydroxycinnaminic acid per ml in acetonitrile-methanol-water (1:1:1) on the target plate. MALDI-TOF-MS spectra were recorded by using a 337-nm nitrogen laser for desorption and ionization. The mass spectrometer operated in the linear mode at an accelerating voltage of 18 kV with an ion flight path that was 0.7 m long. The delay time was 375 ns. Matrix suppression was also used, and the mass spectra were averaged over 50-100 individual laser shots. The laser intensity was set just above the threshold for ion production. External calibration was performed by using the [M + H] + signals of renin, adenocorticotropic hormone, insulin oxidized B, and bovine insulin (Sigma-Aldrich Co.) the results were anticipitated as subtilosin A with m/z of 3400.7 and 3406.6. The variance of the m/z of ±0.8 Da was considered acceptable.

Results
Identification of bacterial strains. Strains were identified according to their morphological and biochemical characteristics added by homology to 16S rRNA with the type strains available in NCBI and RIDOM 35 the results revealed the distinction between two subsp. subtilis and spizizenii as well as their association with their sources.
Detection and sequencing of sbo gene. Sbo and its flanking region were detected from the environmental strain and Type strains. B. subtilis 168 and B. subtilis ATCC 6633 were used as positive control representing the two classes/subsp. subtilis (class I) and spizizenii (class II) respectively. Results are shown in (Table 1) in which majority of strain designated as B. subtilis were secured sbo class I. All the obtained DNA fragments were sequenced, and the phylogenetic relation was established using Clustal W, EBI (Fig. 2).

Detection and isolation and of Subtilosin.
Subtilosin presence was regularly checked on TLC in reference to match the subtilosin produced by B. subtilis 168 further confirmation was carried on using reverse-phase HPLC (Fig. 1A) and MALDI-TOF-MS ( Fig. 1B; Table 1).

Mutation analysis.
Mutants produced further selected based on inhibitory activity. Out of 200 mutant 1 strain was isolated with no detectable zone of inhibition. This (-A) strain was not able to produce subtilosin more over the sbo was not amplified using PCR. Hence, it was determined to be functional disruption of encoding gene (Fig. 3B).

Discussion
It is well established that, the ribosomal peptide antibiotics are synthesized during active growth, while nonribosomal ones are synthesized at later stages. Theories on the role of antibiotic production are yet to be investigated. The best-accepted theory is that nonribosomal antibiotics may play a role in competition with other microorganisms during the starvation phase or spore germination [36][37][38] . While the role of ribosomal peptides remained undefined. Not so obvious the role of such products in the active life cycle. For example the sublancin gene cluster is not essential for B. subtilis, however, it contains yet unidentified genes mediating resistance against sublancin action. Suggestions of an intrinsic mechanism of gene improvement i.e. utilizing antibiotics as first line  www.nature.com/scientificreports/ of defense for survival rather than a second, question antibiotics as secondary metabolites. Another probability is displayed social behaviors as co-ordinate gene expression and group behavior through different quorumsensing pathways 39 . It was determined that interaction of subtilosin with the lipid head group region of bilayer membranes in a concentration dependent manner induced a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers that ultimately resulted in membrane permeabilization at high peptide concentrations 40 . Such adoption may lead to assume a growth control during prosperous stage. Furthermore, under anaerobic conditions an increased by 4-to 90-fold, anticipated that the cell accumulates inactive precursors of subtilosin, which then undergo oxygen-dependent modifications to yield an active peptide when an aerobic environment is encountered 41 . The widespread occurrence of subtilosin might reflect an important physiological role. A specific function of subtilosin as an antibiotic, killing factor 42 or as a pheromone during anaerobiosis 43 or biofilm growth of B. subtilis 39 could be well thought-out. Never the less the gene encoding subtilosin production has demonstrated a strong biomarker for Bacillus subtilis. The B. subtilis strains have segregated into two subclades, one encompassing strain 168 and the other W23, classified strain 168 as B. subtilis subsp. subtilis and W23-related strains as B. subtilis subsp. spizizenii based on DNA reassociation studies 31 and sbo gene analysis 32 . The W23 and 168 group strains are identical for most phenotypic characteristics. However, cell wall chemistry of the W23 strains and 168 strains were different; the cell wall of the former contained ribitol and glycerol teichoic acids and that of the latter only glycerol teichoic acid 44 . The sbo-gene of B. subtilis encodes the 43-aminoacid residue comprising the prepropeptide of subtilosin 23 . The nucleotide sequences of the sbo genes and flanking regions are identical in strains belonging to the same subspecies, and the sequences differ by three nucleotides in the two subspeciesm 45 . However, the encoded Sbo prepeptides are identical in all cases 32 . On the other hand sboX, encoded a bacteriocin-like product, a new gene with an unknown function, in strain 168 23 , which resides in an open reading frame overlapping the coding region of sbo (Fig. 3A). Notably, the expression of sboX would result in a 22-amino-acid curtailed peptide in W23-like strains compared to the peptide produced by 168-like strains, which makes it unlikely that sboX is produced by W23-like strains. These observations were further elaborated to support and to evaluate possible evolutionary relationships among the subtilosin producers, however, A correlation between sbo gene and subtilosin production was not established probably due influence of sboX,. Previous attempts for sboX insertions were not successful as such insertions might render sboA mRNA unstable and explain the reduced subtilosin production in sboX mutant. Alignments has revealed that the sbo genes is highly conserved with those of B. subtilis subsp. subtilis (96-100% amino acid identity), while the remaining were less conserved (83-88% identity). This high and low level of conservation is unprecedented too; for example, thymidylate synthases A (thyA) in B. subtilis subsp. spizizenii ATCC 6633 and W23 and B. subtilis subsp. subtilis (168) exhibit more than 95% amino acid identity 46 . Even the average level of amino acid identity for the DNA gyrases (gyrA) in seven Bacillus type strains was 95.1% 28 . With these results, we can confirm subtilosin gene predominance amongst Bacillus strains isolated from environment and the correlation amongst different sub-species containing homologous genes. Furthermore, this article demonstrated the possibility to utilize Sbo conserved region as a mean of identifying Bacillus spp. that produce subtilosin.